1. Field of the Invention
The present invention relates to a method for detecting alternative splicing in a multicellular organism, a method for identifying substances and gene regions that affect alternative splicing in a multicellular organism, and the like, which utilize a new alternative splicing reporter system developed by the present inventors.
2. Description of the Related Art
Alternative splicing of pre-mRNAs enables multicellular organisms to create a huge diversity of proteomes from a finite number of genes. Many alternative splicing events have been shown to be regulated in cell-type-dependent and/or developmentally regulated manners. However, extensive studies in vitro or in cultured cells have not fully elucidated the regulation mechanisms that determine the specific splicing patterns in living organisms.
The importance of the alternative splicing of pre-mRNAs on the structure and function of proteins, as well as on cellular processes, has been well discussed (Black, D. L. Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367-370 (2000).; Maniatis, T. & Tasic, B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418, 236-243 (2002).; Stamm, S. et al. Function of alternative splicing. Gene 344, 1-20 (2005).). Recent global studies on cDNA sequences or microarray data have predicted that as many as two thirds of human genes have multiple isoforms of mature mRNAs (Modrek, B. & Lee, C. A genomic view of alternative splicing. Nat Genet. 30, 13-19 (2002).; Eyras, E., Caccamo, M., Curwen, V. & Clamp, M. ESTGenes: alternative splicing from ESTs in Ensembl. Genome Res 14, 976-987 (2004).; Kampa, D. et al. Novel RNAs identified from an in-depth analysis of the transcriptome of human chromosomes 21 and 22. Genome Res 14, 331-342 (2004).), and the utilization of alternative splicing microarrays revealed that many alternative splicing events are controlled in tissue- and cell-type and/or developmental-stage dependent manners (Johnson, J. M. et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141-2144 (2003).; Pan, Q. et al. Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform. Mol Cell 16, 929-941 (2004).). These facts indicate that unidentified “cellular codes” underlie the regulation of alternative splicing of so many genes in living organisms (Sharp, P. A. The discovery of split genes and RNA splicing. Trends Biochem Sci 30, 279-281 (2005).; Shin, C. & Manley, J. L. Cell signalling and the control of pre-mRNA splicing. Nat Rev Mol Cell Biol 5, 727-738 (2004).; Hagiwara, M. Alternative splicing: a new drug target of the post-genome era. Biochim Biophys Acta 1754, 324-331 (2005).; Matlin, A. J., Clark, F. & Smith, C. W. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6, 386-398 (2005).; Fu, X. D. Towards a splicing code. Cell 119, 736-738 (2004).). Experimental elucidation of the expression profiles and regulation mechanisms of alternative splicing would lead to a better understanding of genome functions and the cellular identities of multicellular organisms.
Regulation mechanisms of alternative splicing have been experimentally studied mostly in vitro and in cultured cells (Blencowe, B. J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci 25, 106-110 (2000).; Hastings, M. L. & Krainer, A. R. Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13, 302-309 (2001).). General cis-acting enhancer and silencer elements and trans-acting factors, involved in the regulation of both constitutive and alternative exons, have been well characterized by analyzing model genes. Expression cloning strategies have enabled the global collection of putative sequence elements that function in cultured cells and bioinformatic analyses have identified putative cis-elements within exons and introns.
Recently, however, conditional knockouts of trans-acting SR protein families revealed that alternative splicing of only a few target genes are crucially dependent on a specific protein in cardiac muscles, even though many more genes expressed in this tissue have typical cis-elements (Xu, X. et al. ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell 120, 59-72 (2005).). This indicates that we cannot precisely predict the alternative splicing patterns in each tissue or cell without the assessment of the regulation mechanisms in living organisms.